The trace element copper (Cu) is essential for normal growth and development due to its role in a wide variety of biochemical reactions that carry out processes including energy generation, connective tissue maturation, neuropeptide processing, blood clotting, iron distribution and oxidative stress protection. Dietary Cu deprivation or diseases of Cu homeostasis result in impaired motor function, severe cognitive disorders, embryonic and neonatal developmental abnormalities, cardiac hypertrophy, defective angiogenesis and reduced aortic elasticity, anemia and many other pathophysiological states. While a number of genes and their corresponding proteins have been identified in yeast and mammals that function in Cu uptake and distribution at the cellular level, there are many gaps in our knowledge of the function of these components in mammalian physiology. This proposal describes avenues of investigation, using mice as a model mammalian system, to decipher the mechanisms of action and physiological roles of two mammalian Cu transport proteins, Ctrl and Ctr2. In the first specific aim genetic, cell biology and biochemical experiments are outlined to understand the physiological functions of the Ctrl Cu importer in dietary Cu acquisition and in systemic Cu distribution. The second specific aim outlines experiments to dissect the function of the Ctr2 Cu transporter, which localizes to intracellular vesicular compartments, in intracellular Cu transport at the cellular level and its role in mammalian physiology. Taken together, these studies will address critical gaps in our knowledge on the regulation and physiological function of the Ctrl and Ctr2 Cu transport proteins in driving Cu-dependent biochemical reactions that are critical for normal mammalian growth and development.